Evaluation of Shear Bond Strength of Total- and Self-etching Adhesive Systems after Application of Chlorhexidine to Dentin Contaminated with a Hemostatic Agent.

STATEMENT OF THE PROBLEM
Hemostatic agents may influence the bond strength of different bonding agents. Also, chlorhexidine has shown positive effects on bond strength values and their combination effect has not been reported yet.


PURPOSE
The aim of this study was to evaluate the effect of contamination with a hemostatic agent on shear bond strength (SBS) of total- and self-etching adhesive systems and the effect of chlorhexidine application after removal of the hemostatic agent.


MATERIALS AND METHOD
In this experimental study, the occlusal enamel of each sixty caries-free mandibular molars was removed and their midcoronal dentin was exposed. The specimens were then mounted in auto-polymerizing resin 1mm apical to CEJ. Then, the specimens were divided into 6 groups (n=10) based on contamination with a hemostatic agent (H), application of chlorhexidine (CHX) and the adhesive system used; and then were classified as Group 1: Adper Single Bond (ASB); Group 2: H+ASB; Group 3: H+0.2% CHX+ASB; Group 4: Clearfil SE Bond (CSB); Group 5: H+CSB; Group 6: H+0.2% CHX+CSB. Then, composite resin rods (4×2 mm) were built up on the dentin surfaces and after thermocycling, the SBS (MPa) was evaluated. Statistical analysis was performed using two-way ANOVA and post hoc Tukey tests (p< 0.05).


RESULTS
There were statistically significant differences between bond strength values of group 1 (ASB) and group 2 (H+ASB) (p< 0.001) and group 1 (ASB) and group 3 (H+CHX+ASB) (p< 0.001). Similarly, significant differences were seen between group 4 (CSB) and group 5 (H+CSB) (p< 0.001) and between group 4 (CSB) and group 6 (H+CHX+CSB) (p< 0.001).


CONCLUSION
Contamination with hemostatic agent reduced the SBS of both total- and self-etching adhesive systems. In addition, application of chlorhexidine after the removal of hemostatic agent had a negative effect on SBS of total- and self-etching adhesive systems.


Introduction
Good adhesion to tooth structure is one of the prime prerequisites for tooth-colored restorations. Moisture control is essential in order to achieve a durable bond. [1][2] However, isolation against contaminants like saliva, sulcular fluid, blood, and handpiece lubricants, espe-cially near the gingival margins or proximal boxes is difficult. [3] Contamination with saliva and blood can reduce the dentin bond strength of total-and self-etching adhesive systems. [4][5][6][7] A thin film formed by blood proteins and macromolecules such as fibrinogen and platelets impairs adhesion to dentin. Long-term contamination with saliva also causes the formation of a mucin protein layer that impairs the bond. [8][9] Larger particles can be eliminated by rinsing but a reaction between the exposed collagen fibrils and the blood components may inhibit infiltration of primer into dentin. [8] In clinical situations that we have blood contamination, we can use hemostatic agents which are substances that precipitate proteins and are applied to hemorrhagic gingival tissues before placing restorations. [10][11] There are several kinds of hemostatic agents, such as aluminum chloride with a concentration of 5-25%. Having minimal systemic effects, this hemostatic agent is one of the most frequently used astringents that can precipitate proteins, constrict blood vessels, and extract fluid from tissues.
[10] Hemostatic agents are highly acidic and can remove the smear layer and cause some degree of demineralization and their long-term application can even remove peritubular dentin. [11] The effect of hemostatic agents on bond strength has been evaluated in some studies with conflicting results. Mostly, they have shown negative effects on bond strength values. [12][13] On the other hand, it has been shown in some studies that chlorhexidine has a positive effect on shear bond strength (SBS) of total-and selfetching adhesive systems. [14][15] Generally, bond degradation occurs because of resin elution and alteration of collagen fibrils. Exposed collagen fibrils in the hybrid bond layer, not completely infiltrated by resin, are susceptible to degradation that is promoted by matrix metalloproteinase (MMP) enzymes in the saliva and dentin. [16][17][18] MMPs are a host cellderived proteolytic enzyme family with a major role in tissue-destructive inflammatory diseases; they are responsible for degrading most of the extracellular matrix components including different types of collagen. [19][20] Auto-degradation of collagen matrices in incompletely resin-infiltrated dentin can be prevented or minimized with the use of chlorhexidine. [14,21] Its application has led to improved dentin microtensile bond strength after thermocycling, [15] decreasing loss of microtensile bond strength after 2 and 6 years of artificial aging in saliva. [22][23] Functional enzyme analysis of dentin powder also revealed low levels of collagenolytic activity after the use of chlorhexidine. [24] Considering the negative effects of hemostatic agents on bond strength and the positive effects of chlorhexidine, the aim of this study was to evaluate the effect of contamination with a hemostatic agent and application of chlorhexidine after removal of the hemostatic agent on SBS of total-and self-etching adhesive systems. The null hypothesis tested was that contamination with hemostatic agent and application of chlorhexidine after removal of hemostatic agent would not affect the SBS of total-and self-etching adhesive systems.

Materials and Method
In this experimental study, sixty mandibular third molars without any caries, restorations or cracks were collected and used within one month after extraction. Then, the specimens were stored in 0.5% chloramine-T (Fisher Chemical; Fair Lawn, NJ, USA) solution for 24 hours. They were cleaned using an ultrasonic cleaner and used within one month after extraction. The occlusal enamel was removed using a high-speed diamond saw (Leitz 1600; Wetzlar, Germany) under a water coolant and the midcoronal dentin was exposed. Then the teeth were mounted in an auto-polymerizing acrylic resin block (2×2×1 cm) 1mm apical to CEJ and flat dentin surfaces were exposed. The exposed dentin surfaces were further polished with a 600-grit silicon carbide paper for 30 seconds under water irrigation to standardize the smear layer. The teeth were rinsed with distilled water to remove any debris. Then the specimens were randomly divided into 6 groups (n=10) based on contamination with hemostatic agent (Hemostop; Dentsply, Argentina), application of chlorhexidine (Sigma Aldrich, USA), and the adhesive agent chemical composition of aluminum chloride.
In group 1, etching was done with 37% phosphoric acid gel for 15 seconds. After rinsing and air-drying, and Adper Single Bond (ASB) (3M ESPE, USA) was applied as recommended by the manufacturer (Table 1).
In group 2, One drop of hemostatic agent solution (H) was applied directly to the dentin surface for 2 minutes, rinsed for 30 seconds and air-dried. Then the In group 3, One drop of the H was applied directly to the dentin surface for 2 minutes, rinsed for 30 seconds and air-dried. After that, 0.2% chlorhexidine (CHX) solution was applied with a microbrush. The next steps were similar to those in group 1 (H+CHX+ASB).
In group 4, Clearfil SE Bond (CSB) (Kuraray, Japan) was applied to exposed dentin surfaces as recommended by the manufacturer (Table 1).
In group 5, One drop of H was applied directly to the dentin surface for 2 minutes, rinsed for 30 seconds and air-dried. Then, CSB was applied (H+CSB).
In group 6, One drop of H was applied directly to the dentin surface for 2 minutes, rinsed for 30 seconds and air-dried. After that, 0.2% CHX solution was ap-

Results
The means and standard deviations of shear bond strength values are summarized in Table 2. Two-way ANOVA was used to evaluate the interactive effect of adhesive agents and materials (Hemostop and CHX).   The differences between three first groups are shown in different small letters. The differences between three second groups are shown in different capital letters.

Discussion
Hemostatic agents mostly have acidic properties (pH=0.7-3) and hydrophilic characteristics that may affect each stage of the bonding procedure. They cause some changes in dentin surfaces that could affect the hybrid layer quality after the use of total-and self-etching adhesive systems. [11] Thus, penetration of adhesives into dentin surface is prevented and ultimately affects SBS. [3] The first part of this study evaluated the effect of contamination with a hemostatic agent on SBS of both total-and self-etching adhesive systems to dentin. The first part of our null hypothesis indicating that contamination with a hemostatic agent would not affect SBS of total-and self-etching adhesive systems was rejected. A study by Ayo-Yusuf et al. [26] concluded that hemostatic agents can dissolve the smear layer because of their acidic properties and also can obturate the dentinal tubules and demineralize dentinal surface. Granular precipitates, which remain even after acid-etching, were noted on surfaces exposed to aluminum chloride hemostatic agent. The exposure of prepared dentin surface to hemostatic agents altered its morphology and reduced the dentin's susceptibility to acid-etching. [25][26][27] The effect of chlorhexidine application after the removal of hemostatic agent was evaluated in the second part of this study and revealed that use of chlorhexidine after removal of the hemostatic agent reduced the SBS values and the second part of the null hypothe- sis was rejected. Application of chlorhexidine to the cavity prior to its restoration has been recommended in some studies. [27][28][29][30] Residual bacteria have been shown to proliferate from the smear layer even when there is good seal from the oral cavity [27] and this problem may be magnified by microleakage of composite restoration at the margins not ending on enamel.
[28] A possible solution to this problem is treating the cavity preparation with chlorhexidine which has a broad spectrum of action; generally Gram-positive bacteria are more susceptible than Gram-negative ones, particularly Streptococcus mutans that seems to be more sensitive to chlorhexidine. [29] Currently, chlorhexidine is not considered only as an anti-microbial agent, but a potential adjuvant to establishing a better bond to dentin. [30] However, further studies are required in this repute since there is not a standard consensus and some studies have reported different results after the use of chlorhexidine. [30][31] Regarding the step in which chlorhexidine is ap-

Conclusion
Considering the limitations of this in vitro study, contamination with hemostatic agent reduced the shear bond strength of both total-and self-etching adhesive systems. Application of chlorhexidine after the removal of hemostatic agent did not improve the bond.